Analysis of genetic mutations is now rapidly growing as its importance in various fields including human genetic disease detection, pharmacogenetics, drug development, microbiology and the like has been emphasized. In the field of genetics, a mutation refers to a change that occurs in a nucleotide sequence constituting DNA, including insertion/deletion of specific genes such as translocation and inversion, and single nucleotide polymorphisms (SNPs). There among, SNPs are the most general type of alteration in a DNA sequence. In the human genome, SNPs occur at a frequency of every 1,000 bases (Sachidanandam, R. et al. 2001. Nature, 409: 928-933). SNPs occur frequently in non-coding regions rather than in coding regions of the human genome (Li, W. H. and Sadler, L. A. 1991. Genetics, 129: 513-523). SNPs in non-coding regions have been employed as a molecular marker in evolutionary studies, while SNPs in coding regions have been employed as a marker in genetic disease studies and detections since SNPs can affect functions of genes, structures or expression of proteins (Kim, S. and Misra, A. 2007. Annu. Rev. Biomed. Eng., 9: 289-320). Further, various methods capable of rapidly and economically analyzing SNPs as molecular markers have been developed with high reliability and sensitivity (Syvanen, A. C. 2001. Nat. Rev. Genet., 2: 930-942; Kirk, B. W. et al. 2002. Nucleic acids Res., 30: 3295-3311; Kwok, P-Y., 2002. Hum. Mutat., 19: 315-323).
SNP analysis using RT-PCR is most extensively employed and its applicability increases with increasing availability of genetic information. As compared to other SNP analysis methods, RT-PCR has advantages in that RT-PCR is fast, has high sensitivity and specificity, is inexpensive and is easily automated. In addition, unlike conventional PCR, RT-PCR does not require electrophoresis using agarose gels and thus has an advantage of minimizing analysis error due to contamination.
In order to analyze SNPs using RT-PCR, probes or primers in the form of oligonucleotides are used and methods using hybridization probes, hydrolysis probes (or TaqMan probes), molecular beacons, scorpion primers, and the like are mainly used. As a mutation analysis method using hybridization probes, a method employing a Light Cycler PCR system from Roche Co. Ltd., using fluorescent resonance energy transfer (FRET) as a principle has been commercially available (Wittwer, C. T. et al., 1997. Bio Techniques, 22: 176-181). Two probes are used in analysis and FRET uses a principle of generating fluorescence when two probes are in close proximity and thus hybridized with a target DNA. Mutation analysis using hybridization probes may be performed by analyzing melting curves after PCR is completed. Namely, in the case where a partial sequence in the target sequence is mismatched with a probe sequence due to mutation, melting temperatures are lower than the case that the target sequence is completely matched with a probe sequence, which leads to differences in melting curves (Lohmann, S., et al., 2000. Biochemica, 4: 23-28). Methods for analyzing mutations using hybridization probes and melting curves are very rapid and the probes used are relatively easy to design. However, melting curves do not always exhibit expected mutation detection ability.
Hydrolysis probe methods use probes to which a reporter and a quencher are attached at both ends together with primers in PCR, and employ a principle of fluorescent resonance energy transfer. Namely, the principle of fluorescent resonance energy transfer refers to a technique that, when the reporter and the quencher are in close proximity, energy transfer from the reporter to the adjacent quencher occurs so as to prevent detection of fluorescence, and as PCR amplification products are increased, probes bound to the target gene are cleaved by 5′→3′ nuclease activity of Taq DNA polymerases, thereby causing the reporter to fluoresce. In the hydrolysis probe analysis method (TaqMan probe assay), it is very important to search for appropriate conditions that allow probes to bind to the target base sequence and probes to be degraded by nuclease activity. Namely, PCR conditions (thermal profile) that allow primers and probes used in PCR to hybridize to the target sequence and probes to be cleaved simultaneously are important. In order to satisfy these two requirements, two-step PCR is generally employed. Namely, in two-step PCR, the process of denaturation is performed at 95° C. and then annealing and extension are performed simultaneously at 60° C. which is 7-10° C. lower than Tm. If PCR is performed at extremely high temperature, probes are separated (strand-displace) from the target rather than cleaved by Taq DNA polymerase, and thus fluorescence does not increase (Logan, J. et al. 2009. Caister Academic Press). TaqMan™ probes have an advantage of using various fluorescence materials, which allows SNP detection or mutation analysis. However, this method has to use short probes so as to confer specificity of probes, which necessarily lowers Tm values and makes maintenance of a stable annealing state difficult. In order to overcome this problem, there is a drawback of having to use expensive minor groove binder (MGB) probes or locked nucleic acid (LNA) probes (Letertre, C. et al. 2003. Mol. Cell Probes, 17: 307-311). MGB TaqMan probes are similar to general TaqMan probes, but maintain a stable annealing state under PCR conditions since MGB TaqMan probes have minor grove binders added at 3′ end, and accordingly, exhibit a high Tm regardless of their short length (Kutyavin, I. V. et al. 2000. Nucleic Acids Res., 28: 655-661). It is possible to analyze SNPs using amplification refractory mutation system (AMRS) PCR principle with TaqMan probes without using separate modified probes such as MGB probes (Ellison, G. et al. 2010. J. Exp. Clin. Cancer Res., 29:132). However, it is very difficult to identify appropriate PCR conditions capable of distinguishing SNPs through ARMS PCR (Punia P. and S. Aunders. N. http://www.horizonpress.com/pcrbooks).
Molecular beacons and scorpion primers are structured probes including stem-loop structures, show higher specificity than linear probes such as hybridization probes or TaqMan probes, and have excellent ability to recognize mismatches so as to be suitable to discriminate similar sequences or SNPs and alleles. However, considering that it is very difficult to design probes having loop structures, is not easy to obtain desired probes that lead to intended results after general manufacture and examination of various types of probes (Bonnet, G., et al. 1999. Proc. Natl. Acad. Sci. USA, 96: 6171-6176; Broude, N. E. 2002. Trends Biotechnol., 20; 249-256.; Tapp, I. et al. 2000. Biotechniques, 28: 732-738).
The most representative real time PCR is TaqMan analysis, which is a hydrolysis probe analysis method using a 5′→3′ exonuclease activity possessed by Taq DNA polymerases as a basic principle. In 1991, Holland et al., disclosed that specific PCR is identified in real time by 5′→3′ exonuclease activity of Taq DNA polymerase when probes having a base sequence complementary to a template DNA are used. Furthermore, it was confirmed that the probes used are cleaved by 5′→3′ exonuclease activity of Taq DNA without discriminating between probes that are 100% complementary to a template DNA and probes having a non-complementary flap site at the 5′-end (Holland P. M. et al., 1991. Proc. Natl. Acad. Sci., 88: 7276-7280). Thereafter, various RT-PCR techniques were developed using probes modified by fluorescent dyes based on the above method, and are widely used in various applications (Heid, C. A. et al., 1996. Genome Res. 6. 986-994.; Livak K. J. 1999. Genet. Anal., 14:143-149).
In general, DNA polymerases of eukaryotes and archaea include DNA polymerase and DNA endonuclease IV, which is also referred to as FEN1 nuclease (Lieber, M. R., 1997. Bio Essays 19: 233-240), wherein flap endonuclease 1 (FEN1) is known to play an important role in removal of a 5′-flap generated in the course of DNA replication and repair procedures (Rossi, M. L. et al., Chem. Rev. 106, 453-473, Kim, K. et al., 1998. J. Biol. Chem., 273: 8842-8848: Klungland, A. and Lindahl, T. 1997. EMBO J. 16: 3341-3348). Specifically, FEN of eukaryotes is involved in progress of cancers, viral diseases, and the like, which draws growing attention in FEN inhibitors, specifically FEN-1 inhibitors, in order to develop new drugs (Mc Whirter C. et al. 2013. J. Biomol. Screen. 18: 567-75).
Conversely, it is known that, in prokaryotic family A polymerases including DNA polymerases (Taq) derived from Thermus aquaticus, 5′-nuclease and DNA polymeraseare located in different domains in a protein and 5′-nuclease has endonuclease (FEN) activity which removes a 5′-flap while also exhibiting general 5′→3′ exonuclease activity (Lyamichev, V. et al. 1993. Science, 260:778-783).
From the experimental results by Holland et al., it can be found that FEN activity may be employed instead of 5′→3′ exonuclease activity of Taq DNA polymerase when there is a flap site at the 5′-end of the probe used. However, existing methods using TaqMan probes do not discriminate between 5′→3′ exonuclease and FEN activity, which are commonly called 5′-nucleases. Invader assay reported in 2005 (Olivier M., 2005. Mutat. Res., 573: 103-110) proposed a method for detecting SNPs using a property of thermostable FEN enzymes, but the method has not been widely used due to low sensitivity resulting from isothermal reactions instead of thermal cycling signal amplification.